A neuropeptide signal confers ethanol state dependency during olfactory learning in Caenorhabditis elegans

Significance An altered internal state experienced during learning can enhance the specificity of learned information and promote situation-specific recall. Intoxication during training can confer state dependency so that recall requires the same intoxication state. Olfactory learning (OL) in Caenorhabditis elegans can become state dependent during ethanol intoxication. A neuropeptide originating outside of the OL circuit activates a receptor tyrosine kinase within the circuit to signal intoxication during learning to confer state dependency. Surprisingly, intoxication is encoded using a distinct mechanism during recall. State information can be added to existing OL but cannot be removed from state-dependent OL. These observations provide insight into the modulation of learning by alcohol and demonstrate that the effects of intoxication on learning and memory are distinct.

All animals used in behavioral assays were age-matched first day adult hermaphrodites that had been reared for at least two generations in uncrowded, well-fed conditions. Three days prior to testing, populations of animals were age-matched as follows: 5 adult worms were allowed to lay eggs for 3-4 hours on each of 20 seeded 6 cm NGM culture plates, and then removed. The resulting progeny were allowed to develop and were tested on their first day of adulthood. In animals carrying constructs expressing channelrhodopsin or anionic channelrhodopsin, the culture plates were wrapped in aluminum foil to minimize possible effects of ambient light. For these strains, on the day before the SDL assay, L4-stage progeny were transferred to ATRcontaining plates in a light-restricted room.
Chemotaxis Assays. Chemotaxis and olfactory learning (OL, also called olfactory adaptation) assays were performed as described by Colbert and Bargmann (6), with minor modifications as described by Bettinger and McIntire (7). Chemotaxis plates were prepared as follows: 10 mL of assay agar (2% agar, 5 mM KPO4, 1 mM MgSO4, 1 mM CaCl2) was aliquoted into 10 cm petri plates and allowed to dry overnight at room temperature. Immediately before the experiment, plates were dried without lids at 37°C for 1 hour. Plates were weighed after drying and, for ethanol-containing plates, 100% ethanol was added to plates to yield 150 mM or 300 mM ethanol. The plates were sealed with Parafilm and ethanol was allowed to equilibrate into the agar for 1 hour. 1 µL of diluted benzaldehyde (1:200 benzaldehyde:ethanol) was pipetted onto a spot on one side of the plate and 1 µL diluent (100% ethanol) was pipetted onto a spot exactly opposite. To each spot, 1 μL of 1 M sodium azide was added to immobilize worms once they reached the spot. Age-matched first-day adult animals were added to plates after washing twice with S-basal (0.1 M NaCl, 0.05 M KPO4[pH 6], 5 mg/l cholesterol) and once with assay buffer (5 mM KPO4[pH 6], 1 mM CaCl2, 1 mM MgSO4). Between 50 and 100 washed worms were pipetted in 10 µL assay buffer onto each plate at a position equidistant from the odorant and diluent spots and slightly off-center ( Figure 1A). Excess liquid was wicked off the plate using the corner of a Kimwipe. After one hour at room temperature, worms were counted; worms were considered to be at the odorant or diluent spots if they were within 1 cm of the spot. A chemotaxis index (CI) was calculated CI = (the number of worms at the odorant spot -the number of worms at the diluent spot)/the total number of worms on the plate. A high CI (close to 1) indicates the odorant acted as a strong attractant, whereas a lower CI indicates that the odorant was a less effective attractant.
C. elegans extrachromosomal arrays are subject to mosaic loss in each animal. We ensured that our cell-specific expression constructs were not lost from the relevant neurons in experimental animals by scoring chemotaxis assays under fluorescent illumination; only animals with the fluorescent marker were included in the calculation of the CI.
Olfactory Learning (OL) Assays. All OL assays were performed as described by Colbert and Bargmann (6) and modified as described in Bettinger and McIntire (7). Pre-exposure plates were prepared as follows: 10 mL of OL agar (3% agar, 5mM KPO4, 1mM MgSO4, 1mM CaCl2) was aliquoted into 10 cm petri plates and allowed to dry overnight. Immediately before the experiment, plates were dried without lids at 37°C for 1 hour. Plates were weighed after drying and, for ethanol-containing plates, 100% ethanol was added to plates to yield 150 mM or 300 mM ethanol. The plates were sealed with Parafilm and ethanol was allowed to equilibrate into the agar for 1 hour. For benzaldehyde pre-exposure, 1uL of 100% benzaldehyde was pipetted onto each of five solidified agar drops on the lid of the pre-exposure plates. Animals were washed off culture plates with S-Basal, (0.1 M NaCl, 0.05 M KPO4[pH6], 5 mg/l cholesterol) then washed once with S-basal and once with assay buffer (5 mM KPO4[pH 6], 1 mM CaCl2, 1 mM MgSO4), then the single population was divided so that roughly equal numbers of animals were placed on all pre-exposure plates, which were then sealed with Parafilm. Animals were incubated in all pre-exposure conditions for 90 min at room temperature, then washed off plates with S-basal, washed once with S-basal, once with assay buffer, and then each pre-exposure population was divided in half and transferred to the paired chemotaxis plates (i.e. ethanol and no-ethanol or blue light exposure and no blue light exposure) for the chemotaxis assay. All conditions that were compared were performed at the same time such that a single population of worms was divided when placed onto the pre-exposure plates. Table S1. PCR was performed with the Q5 High Fidelity DNA Polymerase (New England Biolabs), DNA sequences were assembled using HIFI DNA Assembly Kit (New England Biolabs), or were generated using BsmI, BamHI, HindIII, or EagI restriction enzymes and ligated using a DNA ligation kit (ThermoFisher). The resulting clones were sequenced to ensure that no mutations were introduced. Extrachromosomal arrays carrying each DNA construct were generated through germline injection and transformation of an appropriate strain (8) and lines with a high transmission frequency for the extrachromosomal arrays were selected for analysis.

Constructs. All primers and sequences used in this study can be found in
pPD95.75 T2A. We inserted T2A (9) downstream of GFP in pPD95.75 (Addgene plasmid 19327). T2A is a self-cleaving peptide that induces ribosome skipping during translation. The T2A sequence was synthesized as a gBlock (IDT) that included vector homology arms, eliminated the stop codon from GFP, and added an NheI site downstream of T2A. pPD95.75 was digested with BsmI and EcoRI and T2A was inserted using DNA assembly.
Optogenetics methods. All animals used in these studies were reared on OP50 bacteria that were supplemented with 10 µM all-trans retinol (ATR; Sigma-Aldrich) 24 hours before the culture was used for seeding bacterial lawns, except for animals that were used as -ATR controls. Animals reared on ATR-supplemented bacteria were kept in the dark. All optogenetic experiments were performed in a dark room with a red light source to minimize any possible activation of channelrhodopsin by ambient light.
To activate channelrhodopsin ChR2, we used a delivery paradigm described by Crawford and San-Miguel (13): exposure consisted of 1-minute bouts made of 30 seconds of 465 nm light flashes [10 milliseconds on; 10 milliseconds off] and 30 seconds of no light; the number of minutes of light exposure varied in the different experiments and is described. We used Matlab (MathWorks) to drive an Arduino circuit board (Arduino) connected to an LED driver (Digikey) which held the current at 350mA; a single pole single throw (SPST) relay closed the circuit to allow a 1.5V LED (Digikey) to 'blink on' and 'blink off'. We measured light intensity at the center of the plate at 0.55 mw/mm 2 and minimally at 0.15 mw/mm 2 at the edge of the plate using a laser power meter (Sanwa).
The anionic channelrhodopsin GtACR1 is sensitive to green light that is in the 460-560 nm wavelength range. Because there are similar effects of GtACR1 on C. elegans behavior at 460 nm and 525nm light (14), we used the same light (465 nm) delivery protocol as described for channelrhodopsin ChR2.
Dopamine supplementation. Dopamine culture plates were prepared by adding dopamine solution (50 mM dopamine hydrochloride (DA; Sigma-Aldrich) in assay buffer) to a final concentration of 2 mM DA to ATR plates seeded with OP50. JCB340: cat-2(e1112); betEx12 [Pgcy-5::ChR2(H134R)::YFP] animals were cultured on these plates for 24 hours before testing as described for optogenetics experiments. For the SDL assay, dopamine was supplemented on pre-exposure plates (2 mM DA, 0.1% metabisulfite in assay buffer). We found that adding DA to the chemotaxis plates caused the animals to stop moving, confounding our ability to score the chemosensory response, and therefore we did not supplement DA during chemotaxis.

Fluorescent microscopy.
Transgenic strains with fluorescent reporters were imaged using a Zeiss Axio Imager A1 microscope outfitted with a 63x Plan Apochromat objective and DIC optics (Zeiss). Images were captured with a Zeiss AxioCam MRm using Zen software (Zeiss).

Data analysis.
Unpaired two-tailed multiple T-test analyses (FDR = 5%) (Prism 9, Graphpad Software) were used to compare the mean CIs of 0 mM vs. 150 mM ethanol exposed chemotaxis assays unless otherwise noted. The multiple T-test analyses take into account multiple testing in determining statistical significance; adjusted p values are presented. Figure S1. slo-1 is required for SDL at 150 mM ethanol but not 300 mM ethanol. (A) slo-1(eg142) null mutant animals do not learn state-dependently when trained and tested on 150 mM ethanol; OL that is learned during ethanol exposure is not dependent on ethanol exposure during testing (compare to N2 results in Fig. 1B). (B) In contrast, when slo-1(eg142) mutant animals are trained on 300 mM ethanol, expression of OL requires the presence of 300 mM ethanol during testing. Pale blue filled circle indicates blue light exposure; purple ring indicates benzaldehyde pre-exposure; yellow indicates ethanol exposure. Error bars represent SEM. Statistical comparisons were made using unpaired multiple T-tests (n=8); bars indicate which data sets are being compared; **** p<0.0001; NS not significantly different. Figure S2. Activation of channelrhodopsin requires all-trans retinol (ATR). betEx12 [Pgcy-5::ChR2::YFP] animals express channelrhodopsin in ASER. Animals were cultured in the presence of (+ATR) or absence of (-ATR) all-trans retinol for 24 hours before the experiment. Animals were pre-exposed to benzaldehyde for 90 min to induce AOL. AOL was observed by testing the chemotaxis response to benzaldehyde (CTx 60'). A low CI (below 0.5) indicates that the animals have undergone AOL and recall their pre-exposure to benzaldehyde. If AOL is statedependent, demonstration of AOL requires the presence of ethanol during chemotaxis. (A) In animals reared without ATR, blue light during pre-exposure to benzaldehyde does not affect the expression of AOL; there is no evidence of state-dependency. (B) In contrast, in animals reared on ATR, optogenetic activation of ASER during pre-exposure to benzaldehyde confers statedependency to AOL such that the expression of AOL is dependent on ethanol exposure during chemotaxis. (C, D) ATR exposure has no effect on AOL in the absence of blue light stimulation. Pale blue filled circles represent blue light exposure; purple ring indicates benzaldehyde preexposure; yellow indicates ethanol exposure. Error bars represent SEM. Statistical comparisons were made using unpaired multiple T-tests (n=8); bars indicate which data sets are being compared; **** q<0.0001; NS not significantly different.   Fig. 6). Pale blue filled circles represent blue light exposure; purple ring indicates benzaldehyde pre-exposure; yellow indicates ethanol exposure. Error bars represent SEM. Statistical comparisons were made using unpaired multiple T-tests (n=8); bars indicate which data sets are being compared; *** q<0.0001; NS not significantly different.